Coronaviruses cause respiratory and gastrointestinal diseases in birds and animals. These RNA viruses are genetically variable and can rapidly evolve to infect humans, sometimes causing severe acute respiratory disease. The coronaviruses are set apart in their assembly and secretion from cells by a strategy involving intracellular virus formation and perhaps novel trafficking routes to the outside environment. Further understanding of these late infection events will identify strategies for therapeutic intervention. We have discovered that a prototype mouse hepatitis coronavirus is inhibited at the assembly and / or secretion stages by very low nontoxic concentrations of a proteasome inhibitor. We hypothesize that the inhibitory mechanism involves ubiquitin depletion from cells, a known effect of proteasome inhibitors, because we discovered that the viral E proteins that are central to virus secretion are ubiquitinated on two lysine residues. Our aims are to determine whether pathogenic human coronaviruses are similarly hypersensitive to proteasome inhibitors and then address whether the ubiquitin conjugation of E proteins is central to coronavirus morphogenesis or expulsion out of cells. Our experiments will specifically evaluate whether the locus of inhibitor and ubiquitin action are at the virus secretion stages and will address the novel hypothesis that ubiquitin modifications direct the trafficking of virus-filled organelles to cell surfaces where the virus cargo is liberated. Our findings will reveal novel cell biological features of vesicle formation and organelle transport and will also inform us about the potential to thwart coronaviruses at late infection stages. PUBLIC HEALTH RELEVANCE: Coronaviruses cause respiratory diseases in humans and domesticated animals and in cases of severe acute respiratory syndrome coronavirus the symptoms can be life threatening. Coronaviruses form in a novel fashion by assembling into organellar vesicles and then expelling from cells when the vesicles fuse to the plasma membrane. As little is known about how this process occurs, the proposal aims to unravel mechanisms of coronavirus assembly and secretion from cells and thereby uncover targets for intervention with this essential infection stage.